Analysis of the Na+/Ca2+ Exchanger Gene Family within the Phylum Nematoda

Na⁺/Ca²⁺ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na⁺/Ca²⁺ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na⁺/Ca²⁺ exchangers are divided into three groups based upon stoichiometry: Na⁺/Ca²⁺ exchangers (NCX), Na⁺/Ca²⁺/K⁺ exchangers (NCKX), and Ca²⁺/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na⁺/Ca²⁺ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na⁺/Ca²⁺ exchangers across the phylum Nematoda. In this analysis we identify Na⁺/Ca²⁺ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na⁺/Ca²⁺ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.     

Calcium absorption by fish intestine: The involvement of ATP-and sodium-dependent calcium extrusion mechanisms

Summary Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia,Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca²⁺ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca²⁺ pump (V _m :0.63 nmol·min^–1 mg^–1,K _m : 27nm Ca²⁺) is calculated to be 2.17 nmol·min^–1·mg^–1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na⁺/Ca²⁺ exchanger with high affinity for Ca²⁺ (V _m :7.2 nmol·min^–1·mg^–1,K _m : 181nm Ca²⁺) is calculated to be 13.6 nmol·min^–1·mg^–1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na⁺/Ca²⁺ exchanger. The high affinity for Ca²⁺ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na⁺/Ca²⁺ exchanger will contribute substantially to Ca²⁺ extrusion in the fish enterocyte. Further evidence for an important contribution of Na⁺/Ca²⁺ exchange to Ca²⁺ extrusion was obtained from studies in which the simultaneous operation of ATP-and Na⁺-gradient-driven Ca²⁺ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca²⁺ transporting cell, in which Na⁺/Ca²⁺ exchange activity with high affinity for Ca²⁺ extrudes Ca²⁺ from the cell.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt

We analyzed the ionic composition of the hemolymph of Porcellio scaber in four different stages of the molt cycle using capillary electrophoresis and calcium selective mini- and microelectrodes. The main ions in the hemolymph were K⁺, Ca²⁺, Na⁺, Mg⁺, and Cl⁻. The values for total calcium obtained by means of capillary electrophoresis and calcium selective minielectrodes did not differ significantly from each other. In situ measurements of the free Ca²⁺ concentration ([Ca²⁺]) by means of calcium-selective microelectrodes indicated that Ca²⁺ is not bound in the hemolymph. During molt the [Ca²⁺] is significantly larger than during intermolt. The [Ca²⁺] increased by 13%, 19% and 18% during premolt, intramolt, and postmolt, respectively. The concentration of the other cations and of Cl⁻ decreased significantly between premolt and intramolt. Thus, the rise of the [Ca²⁺] in the hemolymph is not due to a general increase in all ions, but rather to the resorption of cuticular calcium. Furthermore, the results suggest that K⁺, Na⁺, Mg⁺, and Cl⁻are extruded from the hemolymph during and/or after posterior ecdysis. Accepted: 5 August 1997     

Acclimation to humic substances prevents whole body sodium loss and stimulates branchial calcium uptake capacity in cardinal tetras Paracheirodon axelrodi (Schultz) subjected to extremely low pH

Kinetics of sodium (Na⁺) and calcium (Ca²⁺) uptake were studied in cardinal tetras Paracheirodon axelrodi acclimated to humic substances (HS, 35 mg C l^?1) and low pH (pH 3·72), parallel to analysis of whole body Na⁺ and Ca²⁺ content. This species had a high uptake capacity (J_max) for both Na⁺ and Ca²⁺ in soft, ion‐poor water. The affinity constant (K_m) did not vary significantly among treatments for either Na⁺ or Ca²⁺. J_max Na⁺ increased 30% in fish acclimated to HS for 5 weeks. Acclimation to low pH had no effect on J_max Na⁺ but this treatment was associated with a 32% decrease on whole body Na⁺ content, suggesting that fish were unable to compensate for the increased Na⁺ loss induced by extreme acidity. Exposure of fish to HS + low pH, the treatment most closely approximating to the conditions experienced by the species in its native environment, resulted in an increase in whole body Na⁺ by 31% relative to acclimation to low pH alone. J_max Ca²⁺ in cardinal tetras was high relative to that documented in other freshwater species acclimated to soft water (J_max= 30 nmol g^?1 h^?1). Prolonged exposure of fish to pH 3·72 inhibited J_max Ca²⁺ by 53%, although whole body Ca²⁺ content remained unchanged relative to control. Acclimation of fish to HS + low pH resulted in an increase of J_max Ca²⁺ by 166% relative to low pH alone. Collectively, these results suggest that HS protect cardinal tetras acclimated to soft, acidic waters by preventing excessive Na⁺ loss (as indicated by whole body Na⁺ content) and by stimulating Ca²⁺ uptake (as indicated by increased J_max Ca²⁺) to ensure proper homeostasis.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca²⁻]_i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca²⁻]_i and intracellular pH (pH_i) in Na⁺-containing and Na⁺-deficient bicarbonate/CO₂-buffered media to investigate the role of Na⁺ in these P-initiated changes. We found that P failed to initiate the AR in Na⁺-deficient medium, and that the initial rise in [Ca²⁺]_i following P (1 μg/ml) stimulation was similar for both media; however, the [Ca²⁺]_i in the Na⁺-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca²⁺]_i. Moreover, the differences in plateau [Ca²⁺]_i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca²⁺]_i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca²⁺]_i and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na⁺-dependent mechanism is important in the P-initiated human sperm AR. Such a Na⁺ requirement may reflect the involvement of this ion in pH_i regulation, as capacitated sperm that were incubated in a Na⁺-deficient medium for ≥ 30 min displayed a significantly lower pH_i. © 1996 Wiley-Liss, Inc.     

Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology

Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca²⁺ homeostasis and Ca²⁺ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na⁺, whereas Ca²⁺ can be replaced by Mg²⁺ and Sr²⁺ and that NCX_Mj can be inhibited by divalent ions, particularly Cd²⁺. By directly comparing the apparent affinities of Na⁺ and Ca²⁺ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology.     

披针叶黄华试管苗适应盐胁迫的渗透调节机制

以披针叶黄华(Thermopsis lanceolata)试管苗为材料,通过组培方法研究其在0、0.2%、0.4%、0.6%、0.8%和1.0%NaCl和Na2SO4胁迫30d后的生长、有机渗透调节物质和无机渗透调节物质(Na+、K+和Ca2+)含量的变化,以探讨其耐盐性机制。结果显示:(1)随NaCl和Na2SO4胁迫浓度的增加,披针叶黄华试管苗叶片脯氨酸和可溶性糖含量均显著持续增加,且NaCl胁迫下脯氨酸上升的幅度均大于相同浓度Na2SO4胁迫下的增幅,而可溶性糖上升的幅度却小于相同浓度Na2SO4胁迫下的幅度;可溶性蛋白含量随NaCl浓度的增大呈先升高后降低的趋势,但随Na2SO4浓度的增加呈持续上升的趋势。(2)随NaCl和Na2SO4浓度的增加,披针叶黄华试管苗Na+含量呈增加趋势且各处理均显著高于对照,Ca2+含量和叶片K+含量却呈逐渐减少趋势且各处理均显著低于对照,而根系K+含量呈先降后升的趋势;Na2SO4胁迫下披针叶黄华试管苗叶片Na+含量上升幅度以及K+和Ca2+含量下降幅度均明显低于相同浓度NaCl胁迫组;而Na+/K+和Na+/Ca2+比值随NaCl和Na2SO4浓度增加而升高;NaCl胁迫下,叶片Na+/K+和Na+/Ca2+高于相同浓度Na2SO4胁迫下的比值,而根系Na+/K+和Na+/Ca2+却低于相同浓度Na2SO4胁迫下的比值。研究表明,盐胁迫下,披针叶黄华试管苗通过抑制叶片中Na+积累并增加可溶性糖和可溶性蛋白含量,在根系中维持较高K+和Ca2+含量以及较低水平Na+/K+和Na+/Ca2+比,以降低披针叶黄华细胞渗透势来适应盐渍环境;披针叶黄华对NaCl胁迫的调节能力弱于Na2SO4。     

Searching for a Role of NCX/NCKX Exchangers in Neurodegeneration

Control of intracellular calcium signaling is essential for neuronal development and function. Maintenance of Ca²⁺ homeostasis depends on the functioning of specific transport systems that remove calcium from the cytosol. Na⁺/Ca²⁺ exchange is the main calcium export mechanism across the plasma membrane that restores resting levels of calcium in neurons after stimulation. Two families of Na⁺/Ca²⁺ exchangers exist, one of which requires the co-transport of K⁺ and Ca²⁺ in exchange for Na⁺ ions. The malfunctioning of Na⁺/Ca²⁺ exchangers has been related to the development of pathological conditions in the regulation of neuronal death after hypoxia–anoxia, brain trauma, and nerve injury. In addition, the Na⁺/Ca²⁺ exchanger function has been associated with impaired Ca²⁺ homeostasis during aging of the brain, as well as with a role in Alzheimer’s disease by regulating β-amyloid toxicity. In this review, we summarize the current knowledge about the Na⁺/Ca²⁺ exchanger families and their implications in neurodegenerative disorders.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

Summary The countertransport of Ca²⁺ and Na⁺ across the membranes of the unicellular fresh-water algaChlamydomonas reinhardtii CW-15 and twoDunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by twoDunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na⁺ release. For 15 and 30 s after artificial gradient formation (Na_int ⁺ greater than Na_ext ⁺) the ratio of released Na⁺ to absorbed Ca²⁺ was 31 and 41, respectively. For the extremely halotolerantD. salina, the apparent Michaelis constant of the Ca²⁺ uptake was 33 M, and for the marine halotolerant algaD. maritima, it was equal to 400 M, presuming more efficient Na⁺-for-Ca²⁺ exchange inD. salina cells. Ouabain, an inhibitor of Na⁺/K⁺-ATPase, suppressed Na⁺ transfer by 25%, whereas the agents blocking Ca²⁺-channels did not affect the transport of Ca²⁺ and Na⁺. The oppositely directed transmembrane Ca²⁺ and Na⁺ transfer was shown to depend on the external concentrations of Na⁺ and H⁺. In the fresh-water algaC. reinhardtii CW-15 (Na_ext ⁺ greater than Na_int ⁺), the direction of Ca²⁺ and Na⁺ fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na⁺-Ca²⁺ exchanger function in plasma membranes of algal cells.     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Novel function of the skin in calcium metabolism in female and male chickens (Gallus domesticus)

To study the role of the skin in differential calcium metabolism in White Leghorn chickens, we compared the composition of suction blister fluid (SBF) collected from cutaneous blisters with blood and serum in female and male animals in various physiological states. As an estimate for interstitial fluid (IF), SBF was used as a determinant of local cutaneous metabolism. Sample collection was carried out under ketamine–xylazine anesthesia. Eight chickens of both sexes were raised freely in similar environmental conditions and fed with similar food during their growth from juvenile to sexually mature and fully adult state. SBF, blood and serum were examined for concentrations of ionized Ca²⁺, Na⁺ and K⁺ with ion-selective electrodes (ISEs), and osmolalities by freezing point osmometry. pH and total protein content were also assessed. Our results showed that SBF of chickens is calcium-poor at the juvenile state and that it draws more Ca²⁺ in adult males than laying females of the same age. Interestingly, Ca²⁺ accumulation was observed also in females after laying had ceased. There was a positive correlation between blood and SBF Ca²⁺ in females but a negative one in males. In general, it was found that SBF of chickens was rich in Na⁺ and K⁺, was hypertonic compared to serum at the juvenile state and had a protein content of 36–47% of that in serum. Different from mammals, SBF in adult chickens was alkaline with the mean values of 8.7 ± 0.14 in females and 8.8 ± 0.06 in males. Age- and sex-related variability in cutaneous Ca²⁺ concentrations in chickens, and the differences of SBF composition between that of mammals point to a novel role of skin functions in avians. Possible functions of the skin as a dynamic calcium source balancing the free circulating Ca²⁺ levels and, also, as an excretory organ for Ca²⁺ are discussed.     

Na---Ca exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca²⁺ as essential activator of the Na⁺---Ca²⁺ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 μM free calcium exhibited a 2-fold increase in the initial rate of Na_i⁺-dependent Ca²⁺ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca²⁺ on the reverse mode of Na⁺---Ca²⁺ exchange (i.e. Na_i⁺---Ca₀²⁺ exchange) is saturated at about 100 μM Ca_i²⁺ and displays an apparent K_1/2 of 12 μM. Intravesicular Ca²⁺ produced activation of Na_i⁺---Ca₀²⁺ exchange activity rather than an increase in Ca²⁺ uptake due to Ca²⁺---Ca²⁺ exchange. The presence of Ca_i²⁺ was essential for the Na_i⁺-dependent Na⁺ influx, a partial reaction of the Na⁺---Ca²⁺ exchanger. In fact, the Na⁺ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca_i²⁺ an additional Na⁺---Na⁺ exchange was measured. The results suggest that in nerve membrane vesicles Ca²⁺ at the inner aspect of the membrane acts as an activator of the Na⁺---Ca²⁺ exchange system.     

Calcium Inhibits Paracellular Sodium Conductance through Claudin-2 by Competitive Binding

Claudins form paracellular pores at the tight junction in epithelial cells. Profound depletion of extracellular calcium is well known to cause loosening of the tight junction with loss of transepithelial resistance. However, moderate variations in calcium concentrations within the physiological range can also regulate transepithelial permeability. To investigate the underlying molecular mechanisms, we studied the effects of calcium on the permeability of claudin-2, expressed in an inducible MDCK I cell line. We found that in the physiological range, calcium acts as a reversible inhibitor of the total conductance and Na⁺ permeability of claudin-2, without causing changes in tight junction structure. The effect of calcium is enhanced at low Na⁺ concentrations, consistent with a competitive effect. Furthermore, mutation of an intrapore negatively charged binding site, Asp-65, to asparagine partially abrogated the inhibitory effect of calcium. This suggests that calcium competes with Na⁺ for binding to Asp-65. Other polyvalent cations had similar effects, including La³⁺, which caused severe and irreversible inhibition of conductance. Brownian dynamics simulations demonstrated that such inhibition can be explained if Asp-65 has a relatively high charge density, thus favoring binding of Ca²⁺ over that of Na⁺, reducing Ca²⁺ permeation by inhibiting its dissociation from this site, and decreasing Na⁺ conductance through repulsive electrostatic interaction with Ca²⁺. These findings may explain why hypercalcemia inhibits Na⁺ reabsorption in the proximal tubule of the kidney.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin

Summary Activators of protein kinase C (PKC) stimulate Na^– transport (J _Na) across frog skin. We have examined the effect of Ca²⁺ on PKC stimulation ofJ _Na. Both the phorbol ester 12-O-tetradecanoylglycerol (DiC₈) were used as PKC activators. Blocking Ca²⁺ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na⁺ channels, since the stimulations produced by blocking Ca²⁺ entry and adding cyclic AMP were simply additive.The Ca²⁺ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca²⁺ on PKC or an indirect action on the final receptor site (the Na⁺ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca²⁺, and in fact was inhibited at millimolar concentrations of Ca²⁺.We conclude that the effects of Ca²⁺ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca²⁺ may have stimulated Na⁺ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na⁺ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca²⁺ independent and seems related to thenPKC or PKC observed in other systems.     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.